biology

Review
Mucociliary Respiratory Epithelium Integrity in Molecular
Defense and Susceptibility to Pulmonary Viral Infections
Adivitiya 1 , Manish Singh Kaushik 1 , Soura Chakraborty 1 , Shobi Veleri 2 and Suneel Kateriya 1, *

1 Laboratory of Optobiology, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India;
adivitiya1989@gmail.com (A.); manishskaushik@jnu.ac.in (M.S.K.); sourachakraborty.02@gmail.com (S.C.)
2 Drug Safety Division, ICMR-National Institute of Nutrition, Hyderabad 500007, India;
shobi.veleri@icmr.gov.in
* Correspondence: skateriya@jnu.ac.in

Simple Summary: Mucociliary clearance constitutes an innate lung defense mechanism that is
primarily driven by ciliated cells. Respiratory mucus traps pathogens entering the airways, and
lung cilia propel them outward via their coordinated directional motion. Thus, damage to the
component(s) of this apparatus will hamper its smooth functioning. Here, we update the cellular
and molecular machinery that constitutes and regulates mucociliary clearance (MCC). We also
describe several respiratory diseases arising due to genetic or acquired molecular shortcomings in the
MCC. The past few decades have seen the emergence of novel viruses that inflame and damage the
respiratory tract. Coronaviruses have been observed to disrupt the ciliated epithelium and abolish its
integrity. Bearing in mind the havoc created by the ongoing pandemic, we outline the significance of
the ciliated respiratory epithelium in defense against such microbial infections. We have predicted
protein interaction networks depicting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-





 2)-manifested implications on the molecular machinery regulating mucociliary clearance. Several
proteins involved in the network were found to interact with SARS-CoV-2 viral proteins upon host
Citation: Adivitiya; Kaushik, M.S.;
infection. This review also emphasizes the importance of the proper management and surveillance
Chakraborty, S.; Veleri, S.; Kateriya, S.
of respiratory health in the elderly and patients with chronic respiratory diseases so that they do not
Mucociliary Respiratory Epithelium
Integrity in Molecular Defense and
bear the impact of a severe or lethal infection.
Susceptibility to Pulmonary Viral
Infections. Biology 2021, 10, 95. Abstract: Mucociliary defense, mediated by the ciliated and goblet cells, is fundamental to respiratory
https://doi.org/10.3390/biology fitness. The concerted action of ciliary movement on the respiratory epithelial surface and the
10020095 pathogen entrapment function of mucus help to maintain healthy airways. Consequently, genetic or
acquired defects in lung defense elicit respiratory diseases and secondary microbial infections that
Academic Editor: Nader Rahimi inflict damage on pulmonary function and may even be fatal. Individuals living with chronic and
Received: 28 November 2020 acute respiratory diseases are more susceptible to develop severe coronavirus disease-19 (COVID-19)
Accepted: 21 January 2021 illness and hence should be proficiently managed. In light of the prevailing pandemic, we review the
Published: 29 January 2021
current understanding of the respiratory system and its molecular components with a major focus
on the pathophysiology arising due to collapsed respiratory epithelium integrity such as abnormal
Publisher’s Note: MDPI stays neutral
ciliary movement, cilia loss and dysfunction, ciliated cell destruction, and changes in mucus rheology.
with regard to jurisdictional claims in
The review includes protein interaction networks of coronavirus infection-manifested implications
published maps and institutional affil-
on the molecular machinery that regulates mucociliary clearance. We also provide an insight into the
iations.
alteration of the transcriptional networks of genes in the nasopharynx associated with the mucociliary
clearance apparatus in humans upon infection by severe acute respiratory syndrome coronavirus-2.

Keywords: mucociliary clearance; lung cilia; goblet cells; mucus; respiratory diseases; microbial
Copyright: © 2021 by the authors.
infections; coronavirus
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
1. Introduction
creativecommons.org/licenses/by/ Life depends on the availability of oxygen in humans. The human respiratory system
4.0/). is an intricate interface for breathing and gaseous exchange. Coupled with the circulatory

Biology 2021, 10, 95. https://doi.org/10.3390/biology10020095 https://www.mdpi.com/journal/biology

Biology 2021, 10, 95 2 of 37

system, it ensures a constant oxygen supply to the living tissues and removes carbon di ox-
ide to properly sustain metabolic homeostasis. The air is filtered, warmed, and humidified
during inhalation before traversing the trachea and the dichotomous respiratory airways.
This air stream comes close, within one cell layer barrier, to the blood stream and thereby
exposes the body, via the respiratory tract, to a myriad of foreign agents like pathogenic
bacteria, viruses, gaseous, and particulate matter present in the inhaled air. However, the
respiratory tract has sophisticated defenses to guard against these potentially noxious
agents. Mucociliary clearance (MCC) is the innate lung defense machinery used to capture
and clear inhaled foreign agents [1,2]. The airway mucosa is lined by a pseudostratified ep-
ithelium where the ciliated and secretory cells provide the primary barrier during attack by
a foreign agent [3,4]. The mucosa is covered by the airway surface layer (ASL). It includes a
protective layer of mucus to ensnare the inhaled foreign elements and microbes, as well as a
periciliary layer (PCL) for the lubrication of respiratory airways to facilitate efficient ciliary
movement that drives effective mucus expulsion. In conjunction, the metachronal beating
of cilia on the epithelial surface generates a wave-like motion that propels the pathogens
and particulates trapped within the mucus layer of the airways outward and towards
the nose or mouth for their elimination via coughing or swallowing. Thus, in healthy
individuals, an effective MCC system coordinates the mucus formation (that traps dust
and pathogens to be propelled out) and the clearance of foreign agents mediated by cilia.
Consequently, a weakened or collapsed defense due to the malfunctioning of one or more
components of the MCC apparatus permits the development of chronic respiratory diseases
that may be genetic or acquired, such as chronic obstructive pulmonary disease (COPD),
asthma, cystic fibrosis (CF), and primary ciliary dyskinesia (PCD). Furthermore, smoking,
air pollution, and the inhalation of dust or chemical particles (occupational hazards) also
increase the risk for developing respiratory infections and airway diseases, thereby con-
tributing to the global burden of pulmonary diseases [5,6]. Additionally, socio-ecological
changes in the current century have resulted in the emergence of virulent pathogens, such
as the coronaviruses, that target the respiratory tract. Among them, the infection by severe
acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has evolved into the pandemic
coronavirus disease-19 (COVID-19). The current situation poses an enormous challenge for
managing the pulmonary diseases with an unprecedented dimension. In this context, we
review the important components of the human respiratory system with a specific focus
on the MCC apparatus, highlighting the critical role of cilia and mucus to effectively clear
pathogenic agents entering the lungs. We also discuss the health hazards associated with a
dysfunctional mucociliary escalator. Finally, we present an insight into the impact of SARS-
CoV-2 infection on molecules (proteins) associated with or involved in the mucociliary
clearance system of the host.

2. Organization and Components of the Human Respiratory System

The respiratory system is a complex arrangement of tissues and organs whose primary
function is gaseous exchange. The nostrils, the nasal cavity, and the oral cavity are the
portals for entry of external air into the lungs. However, unlike the oral route, the nasal
route has hair (vibrissae) for trapping unwanted air-borne contaminants. The inhaled air
then journeys en route to the larynx to reach the trachea. The trachea bifurcates into the left
and right bronchi on either side of the chest cavity (thorax). The bronchi enter the lungs at
the hilum and further split into narrower pathways termed bronchioles. These bronchiolar
branches terminate at the alveoli, the sites of large volume of gaseous exchange in our
body (Figure 1a).
Biology 2021, 10, 95 3 of 37

Figure 1. A pictorial visualization of the morphology and development of the components of the respiratory epithelium.
(a) The human respiratory system comprises of the upper and lower respiratory tracts that collectively function to carry out
gaseous exchange and also protect against air-borne infections. The nasal vestibule in the upper respiratory tract is lined by
stratified squamous epithelium (Inset 1). It transitions into a pseudostratified columnar epithelial mucosa with ciliated and
goblet cells lying on a basement membrane (Inset 2). The narrower bronchioles are lined by simple, cuboidal epithelium
with less ciliated and more club cells (Inset 3), while the alveoli are composed of type I and type II alveolar cells (Inset 4).
(b) Development of ciliated and goblet cell lineages. (c) The mucociliary escalator. (d) The 9 + 2 microtubular organization
of a motile lung cilium. Illustration made using BioRender.com.
Biology 2021, 10, 95 4 of 37

The respiratory system is functionally and anatomically divided into two regions. The
route from the nose to the bronchioles that helps in conducting external air to the inner
portions of the lungs is functionally termed the conducting zone, while the portion of
the respiratory system from the alveolar duct to the alveoli is functionally termed as the
respiratory zone. Based on anatomy, the human respiratory system can be divided into the
upper and lower respiratory tracts (URT and LRT, respectively) (Figure 1a) [7].
The anterior portion of the nasal cavity, the vestibule, provides the portal for gaseous
exchange with the external environment and is lined with stratified squamous epithelium
(Figure 1a, inset 1) [8]. Subsequently, the epithelium transitions into a pseudostratified
columnar type with intercalated ciliated and goblet cells (Figure 1a, inset 2) that overlie
a lamina propria carrying muco-serous glands. This region also houses the basal cells,
which can generate the secretory and ciliated cell lineages [5]. Approximately 60% of the
cells lining the trachea are ciliated and 20% are goblet (secretory) cells, and these two
directly contribute to the mucociliary function. Within the smaller airways of the LRT,
the epithelium transitions to a shorter, simple, cuboidal type that is less ciliated and has a
high number of club cells but reduced goblet cells (Figure 1a, inset 3) [7]. Deep within the
lungs, the alveoli are composed of thin type I and type II alveolar cells that are involved in
gaseous exchange and surfactant production, respectively (Figure 1a, inset 4) [9].
The goblet cells are the chief secretory cell type lining the tracheobronchial mucosa.
These adhere to one another by means of tight junctions and are intercalated between the
ciliated cells to form a physical barrier against external contaminants. Goblet cells are
highly polarized with a basal localization of the nucleus and other organelles [7]. The
apical cytoplasm is rich in membrane-bound secretory granules that contain mucin, a high
molecular weight, gel-forming glycoprotein. The serous and club cells within the smaller
airways have small secretory granules that produce watery secretions. The muco-serous
glands in the submucosa supplement the epithelial cell secretions, and, together, they help
in warming and moistening the inhaled air [10]. Together, these secretions constitute a
majority of the airway surface layer.
The ciliated cells of the pseudostratified epithelium are a terminally differentiated
population of epithelial cells with an elongated columnar appearance [11,12]. They are
distinguished by the presence of ~300 cilia per cell on the luminal surface. The cilia have a
diameter of ~0.3 µm and a length ranging from ~7 µm in the upper air tracts to ~4 µm in the
narrower ones [5]. Neighboring cells are inter-connected by means of tight junctions [13].
The apical region of these ciliated cells is rich in mitochondria for sufficient ATP production
that drives ciliary movement triggered by the axonemal dynein motor function [14]. The
ciliated airway cells mediate the propulsion of the tracheobronchial secretion in a cephalad
direction by a coordinated metachronal beating of the cilia [15].
The basal cells function as the progenitor for differentiation into the ciliated cell and
goblet cell lineages. This developmental pathway begins during lung morphogenesis and
is regulated by several transcription factors [16]. The inhibition of the Notch signaling path-
way drives the ciliated phenotype (Figure 1b) [7]. GMNC, a coiled-coil-domain containing
protein of the geminin family, has been identified to be a master regulator for ciliated cell
differentiation that functions downstream of Notch signaling [17]. The transcriptional
activation of GMNC turns on MCIDAS expression, which encodes for multicilin, another
coiled-coil-domain containing protein that is a transcriptional activator of genes required
for basal body production and FOXJ1 (forkhead box J1, a transcription factor), the chief
controller of basal body docking, ciliogenesis, and ciliary movement [18–21]. RFX3, of the
regulatory factor X family, is a transcriptional co-activator of FOXJ1 and helps to induce
the expression of ciliary genes [22]. MYB, another transcription factor, has been identified
to be required for ciliated cell differentiation and acts upstream of FOXJ1 [23]. Interestingly,
MYB upregulation has been observed in airway diseases like COPD.
A sustained Notch activation drives secretory cell differentiation (Figure 1b) [24].
The SPDEF (SAM pointed domain-containing ETS transcription factor) has been found
to be critical for pulmonary goblet cell differentiation in mice. Its overexpression, in vivo,
Biology 2021, 10, 95 5 of 37

was linked to the upregulated expression of genes like FOXA3 (forkhead box A3), AGR2
(anterior gradient 2), and GCNT3 (glucosaminyl (N-acetyl) transferase 3, mucin type) that
are involved in glycosylation and goblet cell differentiation pathways [25]. SPDEF and
FOXA3 overexpression has been observed during chronic pulmonary disorders such as
asthma and COPD. This induces goblet cell metaplasia and mucus over-production, a key
feature of several respiratory illnesses [26,27].

3. Structure and Composition of the MCC Apparatus in Humans

For effective MCC function, the cilia on the surface of the ciliated epithelial cells
interact with the ASL. It is composed of the PCL (~7 µm) coated with an overlying layer of
mucus (~2–5 µm) (Figure 1c). It also includes a thin layer of surfactant that spreads mucus
all over the epithelial surface. The PCL aids in the lubrication of airway surfaces that
facilitate ciliary beating [7]. In totality, the ASL is critical for normal ciliary performance
and to maintain healthy airways.
Mucus is a complex, gel-like secretion of a non-static nature. It is predominantly
composed of the mucin glycoproteins that provide the structural framework to the mucus.
It primarily functions within healthy airways to entrap foreign pathogens and particles,
dissolve toxic gases, and assist in their removal from the lungs via an effective and di-
rectional ciliary beating and cough. Thus, the mucus layer possesses a movable function,
and mucus with an aberrant rheological property is a key pathological feature of chronic
airway diseases. In addition, mucus acts as a reservoir of host-protective proteins and
polypeptides, helps in preventing ASL dehydration, and allows for pathogen sequestration
by interaction with carbohydrate ligands. The respiratory mucus contains molecules such
as secretory immunoglobulin A (IgA), defensins, and histatins for host defense [28,29].
The mucins produced by the goblet cells may be secretory or membrane-associated.
These molecules exist in a condensed, dehydrated state within secretory granules until
their discharge, either constitutively or by external factors such as inflammatory cytokines
and growth factors, bacterial components, environmental, and chemical pollutants [30].
The hyper-secretion and accumulation of mucus is a key pathological feature of diseases
like cystic fibrosis, COPD, and asthma that result from dysregulated mucin production
stemming from goblet cell hyperplasia. In asthmatics, an acute airway response pathway
that stimulates the over-production of mucins and bronchospasm prevails, thus resulting
in airway obstruction that can be fatal [31].
Mucins are composed of a mucin domain that is rich in serine and threonine residues
that are O-glycosylation sites. About 70% of the mass of the mucins is contributed by the
carbohydrate groups that impart to it features of superior resistance to proteolysis and the
sequestration of pathogens, along with an ion and water binding function [28]. A subset of
secretory mucins also contains cysteine-rich amino and carboxy termini in addition to the
glycosylated mucin domain. These mucins exist in the polymeric form via the formation of
intra- and inter-molecular disulfide bonds. Any alteration of the organization of mucins
within the mucus layer may result in pathological diseases where the layer’s transport
properties and barrier function may be compromised.
Mucins are encoded by several MUC genes, of which MUC1, MUC3A, MUC3B, MUC4,
MUC12, MUC13, MUC15, MUC16, MUC17, MUC20, and MUC21 code for cell surface-
associated mucins, and seven genes code for the secretory type (HUGO gene nomenclature
committee, https://www.genenames.org/) [32]. Among the secretory mucins, MUC2,
MUC5AC, MUC5B, MUC6, and MUC19 are oligomeric and gel-forming, while MUC7 and
MUC8 are non-polymeric [28]. Normal mucus is a mixture of ~97% water, ~1% salt, ~1%
mucins, and ~1% of other proteins [7,33]. It predominantly contains MUC5AC and MUC5B
mucins with low levels of MUC2 [34–36].
The hydration of the ASL allows for efficient ciliary beating. ASL hydration occurs via
an active transport of ions across the ciliated epithelium. Ciliated cells express the epithelial
Na+ channel (ENaC) at their apical membrane, which allows for Na+ influx and the passive
transport of H2 O/Cl− across the ionic gradient and into the lumen. The regulation of the
Biology 2021, 10, 95 6 of 37

Cl− export occurs via the CFTR (cystic fibrosis transmembrane conductance regulator) and
the CaCC (calcium-activated chloride channel) [37].
The PCL is a gel-like layer composed of the MUC1, MUC4, and MUC16 mucins
and tethered mucopolysaccharides [38,39], which create an efficient lubrication layer for
ciliary beating and also restrict the entry of foreign particulates. Its hydration status is also
maintained via active transmembrane ionic transport [37]. The PCL volume is critical for
effective MCC, as insufficient hydration causes the collapse of the mucus layer and the
entrapment of cilia within the mucus, as seen in the case of cystic fibrosis [40].

3.1. Functional Role of Cilia in MCC

The mucus gel, carrying the entrapped contaminants (i.e., potential lung damagers), is
proximally propelled via ciliary beating, which has been shown to be regulated by a variety
of factors such as progesterone, nitric oxide, and secondary messengers including cAMP,
cGMP, and calcium [41–44]. Normally, cilia have two beat frequencies. The slow frequency
results from its intrinsic axonemal dynein ATPase activity, while a higher frequency arises
due to stimulation by specific signaling molecules. External mechanical stressors can
also modulate cilia beat frequency via the stimulation of ATP release [45]. Normal cilia
beat metachronically with a frequency of 12–15 Hz and propel mucus with a velocity of
4–20 mm/min [5,46]. The total volume of mucus expectorated or swallowed per day is
~30 mL [5]. The cilia come in contact with the mucus layer during the forward stroke and
pass underneath it on the reverse stroke, thereby propelling it forward [40]. Ciliary beat
frequency, function, and number can be altered by particulate matter, airway infections,
drugs, and strenuous exercise [47–49]. Moreover, ageing also reduces ciliary beat frequency,
with cilia cross-sections displaying micro-tubular disarrangement and single central micro-
tubules contributing to an increased incidence of respiratory infections in the elderly [50].
Such findings indicate the critical reputation of cilia in upholding respiratory health.

3.2. Components of the Propeller Machinery of the MCC Apparatus

The key propeller during mucociliary clearance is the cilium. The motile cilia of the
lung are capable of exerting a mechanical force with their coordinated beating to create a
directional flow of respiratory fluid (i.e., mucus) within the airways [5]. In addition, the
sensory bitter taste receptors present on motile cilia increase calcium ion concentration,
thereby enhancing the beat frequency and imparting a chemosensory function to motile
airway cilia [51].
Since cilia play a key role in the MCC apparatus, ciliogenesis is inevitable for an
efficient MCC. Consequently, molecules required for ciliogenesis are also critical for an
error-free functioning of the MCC apparatus. Ciliogenesis begins through the formation of
centrosome-derived basal bodies that travel to the apical cell surface and dock with the
membrane to nucleate the development of numerous axonemal microtubules that form the
structural core of the cilium. The proteins required for ciliogenesis and ciliary maintenance
are transported via the bidirectional transport system discovered in Chlamydomonas termed
as the intraflagellar transport (IFT) machinery [52,53]. The ciliary axoneme protrudes
from the plasma membrane and has a 9 + 2 organization in the motile cilia, where nine
doublet microtubules are arranged in an outer circle surrounding a central core of a pair
of microtubules (Figure 1d) [54–56]. The nine peripheral doublet microtubules possess
outer and inner dynein arms that regulate microtubule sliding and ciliary motion. Outer
dynein arms are present at 24 nm intervals and are critical for beat frequency, while
the inner dynein arms, located at every 96 nm along the axoneme, are responsible for
the wave-like formation during ciliary beating [57]. The dynein arms are linked to the
central pair of microtubules via radial spokes to generate the whip-like motion that drives
mucus expulsion.
Biology 2021, 10, 95 7 of 37

3.3. Molecular Network of the Lung Cilia and MCC Machinery Regulating Mucociliary Clearance
in Humans: A Protein Network Analysis
Multi-ciliated cells are involved in the control of directional fluid flow across epithelial
tissues. In the conducting airways, cilia along with the secretory cells are responsible
for mucociliary clearance, which expels out pathogens from the lung. The coordinated
movement of the airway cilia generates a motive force that drives mucociliary clearance,
which averts the infection of the respiratory tracts by pollutants and infectious microorgan-
isms responsible for several acute and chronic diseases of the lungs. To comprehend the
overall molecular mechanism regulating the mucociliary clearance, we selected 39 proteins
reported to be involved either directly or indirectly in this process by referring to relevant
research papers and performed an in silico protein–protein interaction analysis (detailed
methodology is mentioned in the Supplementary Material, M&M S1). We included both
experimentally validated and predicted protein–protein interactions in our analysis of the
protein network. The network of the selected proteins was made using String version 11
(Academic Consortium 2020), and the output was further analyzed using Cytoscape 3.7.2
(San Diego, CA, USA) by employing the betweenness centrality algorithm [58,59]. The
betweenness centrality algorithm determines the shortest path between each pair of nodes
in a network [60]. The nodes with high betweenness value actually have the determining
hold over the network. The evidence supporting the interactions between selected proteins
have shown the FDR (false discovery rate) values < 0.05, which represent the significance
of the association (Table S1). The clustering of the protein network was performed by
employing the Markov clustering (MCL) algorithm [61]. The constructed network consists
of 39 nodes that belong to different clusters (clusters I–VI) representing proteins associated
with the relevant processes of mucociliary clearance system, i.e., ciliogenesis, IFT machin-
ery, mucins and MCC regulation, ciliary functions, and cell cycle regulation (Figure 2). The
betweenness analysis revealed that eight proteins (CDK1, MCIDAS, SOX2, FOXJ1, CEP164,
Chibby-1 (CBY1), CCP110 and ARL13B) may behave as the principal nodes and control
the whole protein network. CDK1 and FOXJ1 are the main effectors in the network and
regulate many of the output nodes in the network. Additionally, the network analysis
revealed three critical connections (i.e., SOX2-FOXJ1, SOX2-CDK1, and CDK1-CEP164)
that might be critical for maintaining balance in the network. Clusters I and II consist
of proteins involved in ciliogenesis and the IFT system (Figure 2). Cluster I has a basal
body protein, Chibby (CBY), as one of the principal nodes. It is essential for accurate cilia
structure and mediates the localization of the IFT machinery to the ciliated cells of the
airway epithelium, the impairment of which affects mucociliary transport due to defective
ciliogenesis (marked by a drastic reduction in the airway cilia number) and results in
chronic upper airway infection [62,63]. The inactivation of CBY causes the accumulation of
IFT88 along with other IFT-B complex proteins including IFT20 and IFT57 (part of cluster
II) in the distended tips of the cilia [62]. In addition, a distal appendage protein, CEP164, is
crucial for ensuring the proper recruitment of CBY and its associated proteins, FAM92A
and FAM92B, to the base of the cilium in multi-ciliated cells, as well as the recruitment of
IFT components to the multi-cilia [63,64]. The recruitment of CBY is an important step for
the ciliary targeting of small GTPases like RAB8, RAB11, and ARL13b, in the multi-ciliated
cells. The interaction of CBY with Rabin8 (a guanine nucleotide exchange factor for the
small guanosine triphosphatase RAB8) promotes the recruitment of RAB8 and the efficient
assembly of the ciliary vesicles [63]. Another ciliary protein, LRRC56, in conjunction with
IFT88, is involved in dynein transport to the tip of the cilia. It was reported that biallelic
variants of LRRC56 cause chronic respiratory infections due to dyskinetic cilia showing
phenotypes like an abnormal cilia beating pattern and the absence of outer dynein arms
(ODAs) in the distal region of the axoneme [65]. Cluster I also shows CCP110 as a princi-
pal node, which is a distal centriolar protein considered to be an important regulator in
primary cilia assembly and motile ciliogenesis [66–69]. During ciliogenesis, the removal
of CCP110 from the mother centriole is a pre-requisite for ciliation to occur [66]. The
optimal level of CCP110 is regulated at the transcriptional and post-transcriptional levels
Biology 2021, 10, 95 8 of 37

in multi-ciliated cells [70]. In multi-ciliated cells, ciliation is controlled via a conserved

transcriptional cascade, where the inhibition of Notch signaling activates multicilin (MCI-
DAS) followed by the establishment of a ternary complex with E2F-4/5 and Dp1 [18,71].
The ternary complex activates downstream ciliary transcription factors, including RFX2
and FOXJ1, which further regulate the expression of core ciliogenesis genes, while other
cell cycle genes remain off [19,72]. Walentek and co-workers demonstrated the enhanced
CCP110 expression during the inhibition of Notch signaling or the stimulation of multi-
ciliogenesis [70]. E2F4, RFX2, and FOXJ1 were also reported to bind at the transcriptional
start site of CCP110 [70]. Furthermore, the miR-34/449 miRNA-based post-transcriptional
regulation of CCP110 was reported, which controls basal body maturation/docking and
ciliogenesis in multi-ciliated cells [73]. Furthermore, Mercey and co-workers demonstrated
the miR-34/449-dependent modulation of small GTPase (R-RAS) pathways to promote
the assembly of the apical actin network [74]. The apical actin network assembly is consid-
ered a pre-requisite for the proper anchoring of centriole-derived neo-synthesized basal
bodies during later stages of multi-ciliated cell differentiation. Walentek and co-workers
demonstrated that, like CCP110, miR-34/449 expression is also activated by ciliary tran-
scription factors [70]. Multicilin initiates the assembly of centriole in G0, an early step
in multi-ciliated cell differentiation [19]. In the MCIDAS mutant, respiratory epithelial
cells were shown to have a primary ciliary dyskinesia-like phenotype, i.e., they had one
or two cilia on each cell that lacked proteins involved in ciliary motility (DNAH5 and
coiled-coil domain containing protein 39 (CCDC39)) [75]. MCIDAS mutants also lack
FOXJ1-regulating axonemal motor protein, as well as CCNO expression, thus suggesting
that multicilin is the chief regulator of CCNO/FOXJ1 for human multi-ciliated cellular
differentiation [75].

Figure 2. The mucociliary clearance (MCC) regulatory protein network. The principal nodes and paths that drive the
mucociliary clearance regulatory protein network were analyzed using Cytoscape 3.7.2. The protein network analysis was
performed by using the betweenness centrality algorithm where the sizes of the nodes (circles) indicate betweenness. The
color scale ranges from blue–green–yellow to orange, which is indicative of low to high betweenness values, respectively.
The clustering analysis was performed by employing the Markov clustering (MCL) algorithm. The perforated shapes
represent different clusters (I–VI).
Biology 2021, 10, 95 9 of 37

In the network (Figure 2), cluster V showed a cyclin O (CCNO) protein, which has
a regulatory role in deuterosome formation and the amplification of centrioles in multi-
ciliated cells [76]. Mutations in CCNO cause ciliary dysfunctions (RGMC, i.e., reduced
generation of multiple motile cilia in respiratory epithelia) due to impaired deuterosome
formation [76,77]. Deuterosomes act as amplification platforms for the generation of centri-
oles in multi-ciliated cells. In humans, mutations in CCNO are also associated with severe
airway diseases. Wallmeier and co-workers executed a whole-exome sequencing approach
to identify recessive CCNO mutations in several patients with chronic destructive lung
disease as a result of insufficient airway clearance [77]. CCNO functions downstream of
the multicilin (MCIDAS) and directs multi-ciliated cell development. Apart from multicilin
(MCIDAS) and cyclin O (CCNO), GEMC1 (GMNC), a protein known for its role in the regu-
lation of DNA replication, has also been identified to cause mucociliary clearance disorders,
i.e., a reduced generation of multiple motile cilia (RGMC) in humans. Terre and co-workers
demonstrated impaired growth, hydrocephaly with a high penetrance, and infertility in
mice lacking GEMC1 due to defective multi-ciliated cells in the brain, respiratory tract, and
germline [78]. In ciliated epithelia, GEMC1 up regulates key transcriptional regulators of
multi-ciliogenesis, i.e., MCIDAS and FOXJ1, where GEMC1 activity is stimulated by E2F5
and inhibited by geminin [79].
In cluster VI, we observed the presence of CDK1 (cyclin-dependent kinase 1), a ser-
ine/threonine kinase that plays a crucial role in cell cycle regulation. In response to
DNA damage, the cell recruits a protein complex consisting of proteins from the poly-
ADP-ribose polymerase (PARP) family, MRE11, RAD40 and NBS1 to induce cell-intrinsic
checkpoints. This protein complex activates the ATM/CHK2 or ATR/CHK1 pathway, both
of which converge towards cell division cycle 25 (CDC25) phosphatase located upstream of
CDK1/cyclin B. The CDK1 and CEP164 interaction in the network could be explained by
the fact that the ATM/ATR protein kinase phosphorylates CEP164 causing the activation
of CHK1, which inhibits the cyclin-A-dependent activation of CDK1 and thus pauses the
progression of the cell cycle [80]. We also observed the transactivation/transformation-
domain-associated protein (TRRAP; regulator acting upstream of multicilin), which binds
to upstream promoter region of several genes (associated with human ciliopathies) and
regulate multi-ciliated cell differentiation and function [81]. Herceg and co-workers (2001)
demonstrated that TRRAP is necessary for mitotic checkpoint and normal cell cycle pro-
gression. CDK1 activity in the TRRAP mutant was compromised, which resulted in a
failure of mitotic arrest [82]. Another important protein observed in cluster VI was CDC6,
an ATPase that is known to be involved in the recruitment of pre-replicative complexes
(pre-RC) at origins of replication during the G1 phase, as well as having a role in checkpoint
activation and maintenance [83]. During pre-RC formation, CDC6 ATPase regulates the
effective loading of minichromosome maintenance (MCM) proteins along with their associ-
ated factor CDT1 onto the origin of replication, whereas the origin of replication complex
(ORC) ATPase causes the release of each loaded MCM unit from the ORC-CDC6 loading
machine [84,85]. The proteolytic regulation of cellular CDT1 level by SCFSkp2 or Cul4-
DDB1Cdt2 ubiquitin ligases is crucial for MCM loading. Moreover, the inhibitor protein
geminin (GMNN) is also known to be involved in the regulation of CDT1 activity [86].

3.4. Physiological Importance of the MCC Apparatus and Associated Disease

The MCC is a crucial defense mechanism against chronic airway diseases and infec-
tions. A breach in this protective barrier permits colonization and infection by pathogens.
Anomalies of MCC may have a genetic basis or may be acquired defects that compro-
mise the defense mechanism against foreign agents invading the lung (Figure 3). Pro-
longed smoking impairs mucociliary activity with temporary delays occurring after each
smoke [87]. The bronchial epithelium of smokers displays pathological changes induced by
cigarette smoke like hyperplasia, metaplasia, the presence of cells with atypical nuclei, and
cilia loss [88]. Shortened airway cilia in smokers are also associated with a reduced MCC
rate [89]. It was suggested that smoking induces the expression of epidermal growth factor
Biology 2021, 10, 95 10 of 37

(EGF) by the ciliated cells that bind to its receptor (EGFR) on the basal cells. This shifts the
basal cell differentiation towards the squamous phenotype with the downregulation of
ciliogenesis and secretory differentiation genes, as seen in the airways of the smokers [90].
The components of cigarette smoke suppress the expression of genes involved in ciliogen-
esis, and the overexpression of FOXJ1 was found to reverse this effect in vitro [91]. Such
anomalies in the MCC apparatus such as a patchy or generalized loss of cilia, squamous
metaplasia, and the hyperplasia of goblet cells are also found in pediatric patients exposed
to passive smoking [92]. These changes in the nasal mucosa ultrastructure may affect
mucociliary function, and such kids may develop persistent sinus infections with increased
severity associated with prolonged exposure to smoke chemicals. The shortening and
loss of cilia also results from the use of drugs like marijuana and cocaine, as well as a
range of environmental pollutants, thereby posing a significant health risk by impairing
MCC [5,93,94].

Figure 3. A schematic illustration of the genetic or acquired defects in the respiratory MCC apparatus that impair the
clearance mechanism of the respiratory airways and lead to a diseased state. Defective cilia structure, function, or cilia loss
may occur due to genetic (primary ciliary dyskinesia (PCD), ciliary aplasia), acquired (smoke), or viral factors, and they
could manifest in the form of diseases like chronic obstructive pulmonary disease (COPD), asthma, and PCD where an
inadequate mucus expulsion causes its build-up in the airways. During cystic fibrosis, a faulty cystic fibrosis transmembrane
conductance regulator (CFTR) and goblet cell hyperplasia result in the reduction of periciliary layer (PCL) volume and
mucus hyper-secretion, respectively. This causes the cilia to collapse, thereby resulting in inefficient MCC. The accumulated
mucus promotes microbial colonization and the exacerbation of the disease. Microbial infections aggravate the condition
and eventually damage the epithelial lining as a result of ciliated cell shedding. Illustration made using BioRender.com.

COPD is a progressive, chronic, inflammatory lung ailment characterized by em-

physema and chronic bronchitis that blocks the airflow through the respiratory passage,
making it difficult for the patient to breathe and leads to wheezing. It occurs due to long
term exposure to chemical irritants, predominantly tobacco smoke. Such gaseous particles
inflame the bronchial mucosa causing chronic bronchitis characterized by the frequent pro-
duction of cough and mucus. The alveoli are damaged as a result of prolonged exposure to
chemical irritants, thus causing emphysema. COPD patients show even smaller lung cilia
Biology 2021, 10, 95 11 of 37

than the healthy smokers and a sluggish ciliary beating consistent with the presentation of
an impaired MCC [49,95].
Asthma is a major non-communicable chronic condition resulting from the inflam-
mation and constriction of the respiratory tract due to allergen exposure. Patients may
experience wheezing, coughing, and tightness in the chest during an asthma attack. Dunnill
reported the presence of mucus plugs in the bronchial tracts, the shedding of the ciliated
cells of the bronchial mucosa, and the impaired clearance of bronchial secretion during
autopsy of patients with asthma [96]. Histopathological changes observed in the bronchial
biopsy specimens of asthmatic children and adults revealed damage to the epithelial
layer with the loss of cilia, the degranulation of mast cells, and the destruction of ciliated
cells [97,98]. The ciliary ultrastructure showed ciliary defects and dysfunction (dyskinetic
and immotile cilia) with a reduced beating frequency, especially in the severely affected
patients with no significant alterations in cilia length [99]. The Th2 cytokine, interleukin-13
(IL-13), is regarded as a key effector molecule for goblet cell metaplasia, reduction of
cilia beat frequency, eosinophil infiltration, IgE production, mucus hypersecretion, and
bronchial hyper reactivity associated with asthma [100–102]. Human airway epithelial
cells cultured in the presence of IL-13 showed a loss of ciliated cells due to reduced FOXJ1
expression manifesting in basal body and ezrin mis-localization [103]. Polymorphisms in
the kinesin family member, KIF3A, have been identified as novel candidates for childhood
asthma [104].
A rare congenital disorder of the mucociliary apparatus termed as “ciliary aplasia” re-
sults in a drastic reduction in the number of motile cilia in the respiratory epithelium [105].
Patients exhibit symptoms of classical PCD such as recurrent pulmonary infections (along
with rhinitis, cough, sputum production, wheezing, fever, and nasal discharge) and infertil-
ity [106]. In 2014, a research group adopted a whole-exome genome sequencing strategy
and identified that the loss-of-function or missense mutations in the CCNO and MCIDAS
genes (coding for cyclin O and multicilin, respectively) are associated with the “reduced
generation of multiple motile cilia (RGMC),” thus leading to a rare mucociliary clearance
disorder in the airway epithelium [75,77]. During ciliogenesis, cyclin O is essential for
the production of basal bodies. Consistent with its predicted mode of action, in vitro cilio-
genesis experiments have shown the defective generation and localization of centrioles in
CCNO mutant cells. MCIDAS lies adjacent to CCNO on the 5q11 chromosome and is also a
key player in ciliated cell differentiation [18]. Both function in the same pathway upstream
of FOXJ1, and multicilin regulates the expression of both these proteins. Both CCNO and
MCIDAS mutations were found to be consistent with an autosomal-recessive inheritance
pattern and clinically manifested as postnatal respiratory distress and recurrent chronic
infections of the URT and LRT including nasal polyps and sinusitis, rhinitis, otitis media,
bronchiectasis, chronic obstructive airway disease, recurrent pneumonia, and infertility.
A chest CT (computerized tomography) scan showed chronic destructive lung disease
with bronchiectasis and mucus plugging. The magnetic resonance imaging (MRI) of the
brain revealed hydrocephalus in a few patients. Respiratory insufficiency and failure
necessitated lung transplantation and was also associated with mortality in a few cases.
The TEM of respiratory epithelial cells showed lesser number of cilia along with basal body
mis-localization. Mutations in CCNO showed some residual ciliary motility, while it was
completely absent in MCIDAS mutations.
Another congenital disorder that impairs mucociliary clearance is cystic fibrosis. It is
an autosomal recessive genetic disorder resulting from mutations in the CFTR/ABCC7 gene
that encodes for an ABC (ATP-binding cassette) transporter [107]. CFTR functions as an
ATP- and cAMP-dependent Cl− ion channel situated at the apical membrane of the cells
lining the respiratory epithelium [108]. Several mutations have been identified to be associ-
ated with the hyper-secretory disease, but Phe508 deletion is the most common [109,110].
A dysfunctional CFTR within the epithelial cells results in a reduced secretion of Cl− ions,
the dehydration and depletion of the ASL, an increased Na+ absorption, and a 5–10 fold
higher mucin to water ratio with an enhanced viscoelasticity [111,112]. This causes the
Biology 2021, 10, 95 12 of 37

mucus layer above to attach to the ciliated cells below, thereby impairing ciliary beating
and mucociliary clearance (Figure 3). As a result, a thick layer of mucus develops within
the air tracts, thus providing a niche for microbial colonization (Figure 3) [113]. Microbes
trigger episodes of acute inflammation and pulmonary exacerbation responsible for the de-
terioration of lung function. The sputum microstructure is significantly altered by elevated
mucin and extracellular DNA content. A high sialylation of MUC2 and MUC5AC was
detected in the patients suffering from CF [114]. These mucins express highly sialylated
and sulphated Lewis x determinants, which are attachment sites for Pseudomonas aeruginosa,
the microorganism most responsible for morbidity and mortality associated with CF. These
results indicate a co-induction of the expression of mucin genes along with glycosyl- and
sulfo-transferases during airway inflammation. To make matters worse, in neonates with
a CFTR mutation, the ASL has an acidic pH due to faulty bicarbonate secretion, which
reduces its antimicrobial activity [115,116]. These factors contribute to the persistence of
infection and inflammation, thus causing a decline in lung function.

3.5. Ciliopathies and Their Relevance to MCC

Since motile cilia are crucial in establishing mucus flow and clearance within the
respiratory tract, any violation of their orchestrated movement promotes the development
of respiratory disorders due to an ineffective MCC. Approximately 5% of the children
suffering from chronic respiratory infections are diagnosed with a ciliopathy known as
primary ciliary dyskinesia (PCD), which is predominantly an autosomal recessive genetic
trait, although it has also been rarely reported to be autosomal dominant or X-linked [117].
It is a heterogenic disorder of the motile cilia occurring in 1 in 15,000 live births and
arises due to mutations in several genes responsible for cilia structure, function, and
assembly or biogenesis [7,118]. Most of these defects can be grouped under (a) short or
absent outer dynein arms (ODA) and (b) both outer and inner dynein arm (ODA and IDA,
respectively) defects. Some of the common clinical presentations of the disease include
neonatal respiratory distress (in 80% of cases) and recurrent pneumonia, chronic rhinitis,
nasal congestion and bronchiectasis, organ laterality defects, chronic sinusitis, chronic otitis
media, and infertility [119–121]. It is also associated with a significant reduction in cilia
beating frequency, abnormal ciliary wave form, and the absence of mucociliary transport
(Figure 3) [122]. Patients exhibit a retarded airway clearance that allows a longer residence
time for pathogens, thereby resulting in recurrent LRT infections that lead to bronchiectasis
and lung transplantation in the severe conditions [123].
The mutations in the genes, DNAI1 (axonemal dynein intermediate-chain gene 1) and
DNAH5 (dynein axonemal heavy chain 5), encoding the components of the ODA manifest
in the form of immotile or hypokinetic cilia. These were the first genes identified to be
associated with PCD [124,125]. A homozygous mutation of the DNAL1 gene encoding the
outer dynein arm light chain 1 via the alteration of the Asn150 reduced the stability of
the axonemal dynein light chain 1 and was linked to PCD [126]. Other PCD-associated
mutations that cause ultrastructural defects in the outer dynein arm were observed in
DNAI2 and TXNDC3 (thioredoxin-nucleoside diphosphate kinase) [127,128]. DNAH11
mutations, however, showed a hyperkinetic ciliary beating, albeit a normal axonemal
ultrastructure, and were thus identified by genetic testing [129,130]. ODA defects lower or
completely eliminate cilia beating frequency [131]. Other mutations in the ODA docking
complex proteins are those in CCDC151 and CCDC114 that result in outer dynein arm de-
fects that cause ciliary dysmotility and severely reduced ciliary beating [132,133]. Proteins
involved in the assembly of the IDA and the dynein regulatory complex, CCDC39 and
CCDC40, were also mutated during PCD [134,135]. CCDC 40 is required for the axonemal
recruitment of the former and is vital for ciliary motility and the formation of the left–right
axis [135]. HYDIN and RSPH4A are genes that encode the central apparatus and radial
spokes, while RSPH1 and RSPH9 are radial spoke head proteins. Their mutations caused
motility defects and resulted in absence of the central pair of microtubules, resulting in a
rotational motion of cilia [136–139]. A normal beat frequency, albeit an abnormal circular
Biology 2021, 10, 95 13 of 37

motion beat pattern, was associated with milder clinical diseases (with features such as
better nasal nitric oxide levels, reduced incidences of neonatal respiratory distress, delayed
commencement of cough with sputum, and better lung function) in RSPH1 homozygous
mutations [139].
In PCD, disease variation and severity are associated with mutations in specific genes.
The biallelic mutations causing a loss of function of CCDC39 and CCDC40 result in ax-
onemal disorganization, as noted by the absence of inner dynein arms and disordered
microtubules in some cilia. It has been reported that these mutations were associated with
severe lung disease in children [117,140]. The CCDC 39 and 40 complex acts as a molecular
ruler to construct the accurate 96 nm spacing of the doublet microtubules in the ciliary ax-
oneme [141]. CCDC65 mutations have a normal axonemal ultrastructure with hyperkinetic
cilia [142]. Several PCD-associated mutations have been found by comparative genomics
and encode cytoplasmic proteins involved in cilia assembly and protein trafficking. Such
mutations cause ultrastructural abnormalities such as truncated or missing IDAs and
ODAs, thus resulting in immotile cilia or severely impaired ciliary beating. These include
mutations in HEATR2 [143], ZMYND10 [144], SPAG1 [145], LRRC6 [146], ARMC4 [147],
DNAAF1 [148], DNAAF2 [149], DNAAF3 [150], CCDC103 [151], and DYX1C1 [152]. Re-
cently, a mutation in the CFAP57 gene has been identified to be associated with PCD.
This protein is involved in IDA assembly, and its absence resulted in a reduced beating
frequency of cilia with an altered beating pattern [153].
Chivukula and co-workers recently showed the correlation of a loss of function
mutation in the NEK10 (NIMA-related kinase 10) gene to the development of familial
bronchiectasis in an autosomal recessive manner [154]. In the human airways, NEK10
specifically expresses in a ciliated cell-specific fashion and is required for an effective
MCC. Its deficiency was seen to reduce the ciliary length. NEK10 was shown to affect
diverse components of the ciliary proteome such as axonemal dynein and assembly factors,
kinesins, proteins of the IFT machinery, and proteins regulating ciliary length. Studies
in Chlamydomonas reinhardtii determined that cilium length was critical to ciliary beating,
and shorter cilia failed to achieve periodic beating [155]. Thus, NEK10 is essential for
generating ciliary movement in the airway for mucociliary clearance.

3.6. Mucociliary Dysfunctions upon Polymicrobial Infections

The air tracts of patients suffering from respiratory diseases can be chronically inhab-
ited by complex communities of diverse microbes [156–158]. Several microbial pathogens
like Bordetella, P. aeruginosa, Mycoplasma pneumoniae, and Actinobacillus pleuropneumoniae
selectively target and bind to the ciliated cells [159]. These polymicrobial infections are
specific to an individual and are important factors in determining the host–pathogen inter-
action, altering the lung environment apart from influencing the disease progression, the
course of treatment, and the clinical outcome of the disease. P. aeruginosa is the commonly
isolated microbe from the sputum samples of adult patients with PCD, while Haemophilus
influenzae colonization is prevalent till adolescence [160,161]. However, other bacterial
pathogens, such as Staphylococcus aureus, Streptococcus pneumoniae, Ralstonia, Moraxella
catarrhalis, nontuberculous Mycobacteria, and Achromobacter xylosoxidans, have also been
identified in PCD sputum [162–164].
In patients with cystic fibrosis, polymicrobial infections trigger pulmonary exacer-
bations causing irreversible lung damage and increase the incidence of morbidity and
mortality accompanying the disease [165,166]. An accelerated worsening of disease symp-
toms such as shortness of breath and increase in respiratory rate, weight loss and appetite
loss, increased coughing and sputum production, hemoptysis (coughing up blood), reduced
lung function, and an increased neutrophil count are key characteristics of pulmonary
exacerbation [167]. While antimicrobial therapy constitutes an effective component of
pulmonary exacerbation treatment, over time, the lung function declines and requires
transplantation [168,169]. Children with cystic fibrosis have been commonly found to carry
H. influenzae and S. aureus infections within their airways, while the microbial community
Biology 2021, 10, 95 14 of 37

changes to P. aeruginosa or Burkholderia cepacia in adults [170]. The neutrophil elastase and
proteinases produced by P. aeruginosa are cytotoxic, reduce ciliary beat frequency, and cause
epithelial damage, thus contributing to a delayed MCC [171,172]. Furthermore, the reactive
oxygen species produced by the polymorphonuclear leukocytes in response to infection
decrease the beating frequency of respiratory cilia [173]. A sputum analysis from 14 adult
CF patients showed the presence of seven core genera belonging to both aerobic as well as
anaerobic bacteria, namely, Pseudomonas, Streptococcus, Neisseria, Catonella, Porphyromonas,
Prevotella, and Veillonella [174]. The Streptococcus milleri group (SMG) was identified as the
cause of chronic pulmonary infections [175]. The fungal Candida spp., Malassezia spp., and
Aspergillus fumigatus were determined to be predominant in the mycobiome of the sputum
from CF patients, while influenza A/H1N1 and respiratory syncytial virus were prevalent
in throat swabs of CF patients presenting with acute pulmonary exacerbation [157,158,169].
The aggressive antimicrobial treatment of such conditions has led to the emergence of
multiple drug-resistant (MDR) non-tuberculous mycobacteria, the Burkholderia cepacia
group, methicillin-resistant S. aureus (MRSA), vancomycin intermediate S. aureus (VISA),
and Trichosporon spp. as infectious agents in CF patients. In addition, advancements in
molecular profiling have helped in identifying other microbial contributors responsible for
chronic lung infections in CF such as Streptococcus anginosus and rhinovirus [169].
Apart from a genetic or acquired environmental cause, ciliary disorientation and
mucociliary dysfunction may also stem from inflammation as a result of microbial infec-
tions [176,177]. Pulmonary infections like bacterial pneumonia and COPD have often been
observed in the HIV-infected population [178]. It was demonstrated that the bronchial ep-
ithelium can be infected with HIV because it expresses the HIV receptors and co-receptors
such as CD4, CCR5 and CXCR4 [179]. This infection interfered with epithelial cell differen-
tiation and suppressed ciliogenesis. In addition, the TAT protein of the virus was found
to suppress CFTR biogenesis via a TGF-β signaling pathway. HIV-infected patients often
exhibit recurrent sinus infections due to a delayed MCC. The nasal nitric oxide inhibits
bacterial and viral growth in the upper airways and has been reported to be about 21%
lower in HIV-infected individuals thereby contributing to an increased susceptibility to
airway infections [180]. The MTT (mucociliary transport time) in non-HIV control individ-
uals was found to be 7.4 ± 3.7 min as opposed to 11.9 ± 5.9 min in patients infected with
HIV. Disease progression from HIV to AIDS, as well as a history of sinus infections, further
delayed the clearance rates to 13.5 ± 6.8 and 13.7 ± 6.8 min, respectively [181].
The respiratory viruses are a major cause of infections that can inflame and injure the
human airways. Such viral infections also stimulate the development of secondary bacte-
rial infections as a result of the impaired integrity of the mucociliary epithelium [182,183].
Additionally, in individuals with existing airway diseases, viral infections may exacerbate
the existing medical symptoms and may be fatal [184,185]. A predisposition to superinfec-
tions by H. influenza, S. aureus, and S. pneumoniae is well-documented for airways infected
with influenza or respiratory syncytial virus (RSV) [182,183]. Researchers compared the
transcriptomic signatures of respiratory viral infection by influenza, hRSV (human res-
piratory syncytial virus), and hMPV (human metapneumovirus) [186]. In HAE (human
reconstituted airway epithelial) models, the influenza viral peak was attained earlier than
hMPV or hRSV, which corroborated well with changes in the epithelial surface observed
via microscopy. There was significant induction of the pro-inflammatory Th1 response
cytokines IL-2 and IP-10 (interferon gamma-induced protein 10), while RANTES (regu-
lated on activation, normal T cell expressed and secreted), IL-8, IL-6, MIP-1B (macrophage
inflammatory protein-1B), IL-1B, and IL-1RA were induced to a lesser extent. Notably,
hMPV infection did not show a relative increase in RANTES and IL-8, while hRSV did
not show a significant effect on GM-CSF (granulocyte-macrophage colony-stimulating
factor). In all cases, there was a significant downregulation of the ciliogenesis-related genes.
Mucociliary movement was completely eliminated in the case of hRSV infection but was
only reduced in the case of hMPV. The MCC rate was reduced more than two folds in the
case of influenza virus infection in comparison to mock.
Biology 2021, 10, 95 15 of 37

4. Coronavirus Disease Manifestation in the Respiratory System

The viruses of the order Nidovirales are enveloped and possess large, positive sense,
single stranded RNA genomes that are non-segmented and can range up to 26–32 kb [187,188].
Within this order lies the family Coronaviridae and the subfamily Coronavirinae, which
houses the four genera of coronaviruses viz alpha, beta, gamma, and delta [189]. The
coronaviruses (CoVs) are known to infect both animals and humans. Of extreme relevance
to this review are the etiological agents for respiratory tract illnesses in humans.
Among the first described coronavirus strains associated with human infections are
HCoV-229E (alphacoronavirus) and HCoV-OC43 (betacoronavirus) that cause mild URT
illnesses in man. Indeed, along with the later discovered HCoV-NL63 (alphacoronavirus)
and HCoV-HKU1 (betacoronavirus), these viruses are endemic in humans and contribute
to 15–30% of the common colds annually [187] that progress to severe LRT infections in the
case of infants, elderly, and the immunocompromised [189]. However, the occurrence of
the severe acute respiratory syndrome (SARS) during 2002–2003 changed the picture. SARS
was the first epidemic of the 21st century caused by an emerging virus from cross-species
viral transmission (zoonosis) and subsequent human to human transmissions. Originating
in Southern China as an outbreak of pneumonia, the disease soon spread across several
countries and has resulted in 8000 confirmed cases with a mortality of 9.6% [190,191].
SARS-CoV, a betacoronavirus, which possibly originated in bats, was identified as the
etiological agent of SARS [192]. The disease varies in its severity from asymptomatic to mild
influenza-like symptoms, and in extreme cases, the onset of a severe form of disease causes
acute respiratory distress syndrome, respiratory failure, and death. Age and co-morbid
illnesses are key factors that enhance the severity of the disease [193].
The clinical course of SARS follows three phases, namely viral replication, immune
hyper-reactivity, and pulmonary tissue deterioration [194]. During the first phase of
infection, patients develop fever with an increase in the viral load in bodily secretions such
as stool, urine, and respiratory secretions. There is a gradual increase in lung damage
as evident from X-ray and CT scans [195]. Phase 2 is marked by the recurrence of fever,
reduction in viral load, oxygen desaturation (SPO2 ), the development of pneumonia,
and respiratory distress syndrome. In the third phase, a sustained pulmonary damage
with a honeycomb-like appearance of lungs on the CT scan, lung fibrosis, and a lack of
oxygen in blood necessitates the intensive care and ventilation of the patients. Finally, the
patient might enter a fatal stage. Recovered patients show signs of long-lasting pulmonary
damage [196–198].
The most commonly reported symptoms of SARS include fever, dry cough, chills,
myalgia, and malaise [194,199]. Patients report a shortness of breath and a lung CT scan
shows consolidation by the end of the first week. Disease manifestations in other organs
include diarrhea, renal and liver dysfunctions, thrombocytopenia, lymphopenia with a
decrease in CD4+ and CD8+ T cells with thrombo-embolic events, and neuro-invasion
occurring in a few cases [200–207]. There is the activation of Th1 cell-mediated immunity
and inflammatory response characterized by a marked elevation of interferon gamma
(IFN-γ); IL-1, 6 and 12; the neutrophil chemokine IL-8; monocyte chemoattractant protein-1
(MCP-1); and the Th1 chemokine IP-10 [208].
In 2012, the Middle East respiratory syndrome (MERS) caused by the MERS-CoV
(betacoronavirus) was the sixth case of coronavirus to cause human infections, yet again
via an interspecies jump from bats to humans with dromedary camels as an intermediate
host [209–211]. Globally, there are more than 2000 confirmed cases of MERS with a mortality
ratio of 34.4% [212]. Like in the case of SARS, a MERS-CoV infection may vary from mild
symptoms to severe acute respiratory disease and death, especially in the elderly and
patients with underlying co-morbidities like diabetes, hypertension, and renal and cardiac
disease [213–215].
The year 2019 ended in the grip of a previously unknown novel coronavirus termed
SARS-CoV-2 or 2019-nCoV (betacoronavirus), the causative agent of the COVID-19 pan-
demic. Originating in the Wuhan city of China as ‘a pneumonia of unknown origin,’ this
Biology 2021, 10, 95 16 of 37

emerging virus has affected the lives of millions of people globally and has become an enor-
mous threat to the public health and global economy. SARS-CoV-2 constitutes the seventh
member of coronaviruses known to infect humans. The genomic characterization of the
virus indicated a genome of 29.8 kb with similarity to corona virus of bat origin [216,217].
It was found to be similar to SARS-CoV in terms of its transmission and pathogenicity,
and it uses the receptor ACE2 (angiotensin converting enzyme 2) and TMPRSS2 protease
(a type II transmembrane serine protease) for cell entry (Figure 3) [218]. Single-cell RNA
sequencing data showed that these elements that mediate SARS-CoV-2’s entry into the
body are predominantly expressed in the nasal epithelial cells, thus providing a molecular
basis for early stage viral transmission [219,220]. Under the TEM, the viruses showed little
pleomorphism and were mostly spherical with 60–140 nm diameter and spikes of 9–12 nm
in length [221]. The novel coronavirus possesses 16 non-structural proteins (NSPs) and four
structural proteins (S: spike; E: envelope; M: membrane; and N: nucleocapsid) [222]. The S
glycoprotein is involved in host cell invasion and infection. The receptor binding domain
(RBD) of the S protein recognizes and attaches to the ACE2 receptor. The S protein is then
cleaved by TMPRSS2 into two subunits (S1 and S2). S1 is responsible for receptor binding,
and S2 is essential for fusion with the host membrane and cell entry [189]. Both ACE2 and
TMPRSS2 are expressed in the secretory and ciliated cells of the airway epithelium [219,220].
ACE2 is highly expressed in the nasal epithelial cells (URT) and is significantly lower in the
bronchi and lung parenchyma (LRT) [220,223]. Considering the high rate of infectivity of
SARS-CoV-2, alternate mechanisms of cellular entry have recently been deciphered for this
virus, particularly in tissues with a low or absent ACE2 expression. Contrary to SARS-CoV,
the S glycoprotein of SARS-CoV-2 possesses a polybasic sequence at the S1–S2 junction
that can be cleaved by the cellular protease furin [224]. This cleavage reaction generates
the sequence ‘RRAR’ at the S1 subunit’s C-terminus that conforms to the C-end rule and
can bind to neuropilin receptors on the target cells [225]. Neuropilin-1 (NRP1) has been
reported to be a host factor for SARS-CoV-2 entry and potentiates its infectivity [226,227].
Interestingly, the gene expression of NRP1 and 2 was observed to be upregulated in the
lung tissues of patients who died from COVID-19 [228]. The members of the C-type lectin
superfamily, CD209L (L-SIGN) and CD209 (DC-SIGN), can also mediate coronavirus in-
fection by interacting with the RBD of the S glycoprotein [229]. CD209L is prominently
expressed in the epithelial and endothelial cells of the lungs and kidneys. It also interacts
with the ACE2 receptor, indicating their heterodimerization and a co-receptor function to
facilitate viral entry in tissues where both ACE2 and CD209L are co-expressed. CD209L has
two sites for N-glycosylation (Asn92 and Asn361), of which only the former is glycosylated.
The removal of the bulky N-linked glycans from this site resulted in the enhancement of
the CD209L-S protein interaction. Additionally, docking studies have revealed that the
RBD of the S protein houses a site for heparin/heparan sulfate binding that lies adjacent
to the ACE2 binding site [230]. Based on in vitro experiments, Claussen and co-workers
proposed a model of the heparan sulfate-mediated augmentation of SARS-CoV-2 binding
to the ACE2 receptor [230]. Yet another novel mode of host entry and invasion by spike
protein exploiting the CD147 receptor was recently demonstrated [231].
SARS-CoV-2 is less pathogenic than SARS-CoV and MERS-CoV but has shown a
wider spread, thus posing challenges for its mitigation [232]. Fever, cough, and fatigue
are the most commonly reported symptoms for COVID-19; however, sore throat, diarrhea,
headache, sputum production, and shortness of breath have also been reported [233,234].
Patients may exhibit no symptoms or mild to severe symptoms that can be fatal. Clini-
cal studies have shown that individuals with endocrinopathies (diabetes, hypertension,
obesity, and cardiovascular diseases) show increased complications [235] associated with
poor prognosis. Patients suffering from a severe disease also exhibit coagulation disorders,
as observed during SARS-CoV and MERS-CoV infections with high circulatory levels
of D-dimer, fibrin degradation products, and thromboembolic complications [236–239].
Thrombocytopenia, lymphocytopenia, leukopenia, and elevated C-reactive protein levels
have also been observed [240]. Chemosensory dysfunction, such as smell and taste impair-
Biology 2021, 10, 95 17 of 37

ment, has also been accepted to be associated with COVID-19 infection among clinicians
and healthcare workers [241–243].

4.1. Coronavirus-Induced Dysfunctions of the Lung Cilia

In 1994, Afzelius described the ultrastructure of the nasal epithelium upon coronavirus
infection using electron microscopy, wherein virion particles showed a specific affinity
towards the ciliated cells but not the goblet cells [244]. In addition, while viral infection
did not induce the destruction of the ciliated cells, however the retraction of the cilia into
the cell body was observed in a few cells, thus leading to ciliary loss. Such a selective
attachment to ciliated epithelial cells has also been seen in case of other respiratory viruses
like myxoviruses, respiratory syncytial viruses, influenza, and rhinoviruses [245,246]. The
loss of cilia along with diffused alveolar damage, hemophagocytosis, bronchial epithelial
denudation, and squamous metaplasia are key morphological changes that have been
observed in the lungs of deceased patients from SARS [247,248]. Secondary bacterial or
fungal infections were also reported in case of a few autopsies [249].
In a quest to understand the magnitude of coronavirus infection on the destruction of
ciliated respiratory epithelium of the human nose, scientists inoculated 15 healthy adult
volunteers with HCoV-229E via the nasal route. Eight of the 11 exposed to the active virus
developed mild symptoms of URT infection like cold, cough, fever, and headache. Samples
of ciliated epithelium obtained from all 11 test subjects at zero and three days after infection
showed a defined loss of respiratory epithelium integrity marked by a significant reduction
in ciliated cells and an increase in dead cell population. Furthermore, there was an increase
in micro tubular abnormalities on the third day post-infection with ciliary dyskinesia [250].
Such a massive ciliary loss during coronavirus infections may lead to rhinorrhoea [244,251].
The etiological agent of the COVID-19 infection, SARS-CoV-2, has also been linked
to ciliary loss and damage to the pulmonary epithelium (Figure 3). The bronchoalveolar-
lavage fluid specimens obtained from three patients known to frequent the seafood whole-
sale market in Wuhan, the epicenter of the COVID-19 outbreak, were used to inoculate
human airway epithelial cells cultured such that they resembled the pseudostratified air-
way epithelium of the respiratory system. After 96 h of inoculation, the cells exhibited
cytopathy and a lack of ciliary beating under a light microscope [221].
All these morphological evidences outline the damage to the primary defense barrier
of the human respiratory system, the cilia, during coronavirus infections. Ciliary loss
is associated with inefficient mucus and pathogen clearance from the airways. Mucus
build-up will attract secondary microbial infections that can complicate recovery from
disease. Hence, MCC defects like asthma, COPD, PCD, and cystic fibrosis are risk factors
for developing COVID-19. Furthermore, ciliary beat frequency is known to slow down
with age [252], suggesting that the current pandemic is a real hazard for the lives of the
elderly population.
Another key symptom pointing towards ciliary damage during SARS-CoV-2 infection
is anosmia (the loss of the sense of smell) [253–255]. In the human body, such olfactory
functions are linked to the sensory olfactory cilia that detect airborne stimuli and transmit
electrical signals to the olfactory cortex within the brain [256]. Anosmia resulting from
a deficit of proper ciliary structure or function has been observed in a ciliopathy termed
Bardet–Biedl syndrome (BBS) [257]. Hypomorphic mutations of the CEP290 protein causes
Leber congenital amaurosis that manifests as retinal dystrophy and severely aberrant
olfactory function [258]. The interaction of SARS-CoV-2 Nsp 13 (non-structural protein-13)
with centrosomal components may provide a molecular link to COVID-19 infection and
anosmia [222,259].

4.2. SARS-CoV-2-Manifested Implications on the Molecular Machinery Regulating

Mucociliary Clearance
As discussed in the previous sections, mucociliary clearance dysfunction leads to
compromised respiratory health. Considering the harmful impact of SARS-CoV-2 on the
respiratory system, we aimed to comprehend the implications of SARS-CoV-2 infection on
Biology 2021, 10, 95 18 of 37

the molecular machinery regulating mucociliary clearance. To get a clear picture of the cel-
lular players involved and the complex interactions that regulate the mucociliary clearance
following SARS-CoV-2 infection, we selected 36 proteins by referring to relevant research
papers and constructed a protein–protein interaction network (detailed methodology is
present in the Supplementary Material, M&M S1). The FDR values representing the confi-
dence and significance of the protein interactions are mentioned in Table S2. The obtained
network consisted of 36 nodes that belong to different clusters (clusters I–VIII) represent-
ing proteins associated to the mucociliary clearance system relevant processes (Figure 4a).
Clusters I–V consist of proteins involved in cell cycle regulation, IFT machinery, cilia
biogenesis and regulation, cAMP regulation, and immune defense, respectively, whereas
clusters VI–VIII represent proteins that act as SARS-CoV-2 entry point and infection targets,
respectively (Figure 4a). The betweenness analysis revealed that seven proteins (CDK1,
IFT88, FOXJ1, ADCY3, TNF, PPIA, and ACE2) may behave as the principal nodes and
control the whole protein network. IFT88 and TNF are the main effectors and regulate
many output nodes in the network. Cluster I shows an interaction between two cell cycle
regulator proteins, CDK1 and MCM3 (Figure 4a), which are also described as components
of the molecular machinery regulating mucociliary clearance (Figure 2). CDK1 phospho-
rylates Ser112 in MCM3, which results in its incorporation into MCM2-7 complex and
loading of the MCM3 onto chromatin in cycling cells [260]. The MCM2-7 complex (ATP-
dependent helicase) causes the unwinding of DNA and regulates the initiation of DNA
replication as well as replication fork progression in association with ORC and Cdc6 [261].
Furthermore, CDK1 also showed interactions with DYNLL1 (a component of the dynein
light chain) and RAB8A in cluster II (IFT machinery) (Figure 4a). In yeast, CDK1 controls
the fidelity of chromosome segregation by regulating nucleolus spindle kinetochore 1 and
dynein light chain 1 complex (Nsk1–Dlc1) at the kinetochore-microtubule interface [262].
Reports have also demonstrated DYNLL1 interactions with astrin–kinastrin/SKAP (small
kinetochore-associated protein) and human NSK1-like proteins involved in controlling
proper chromosome movements [263,264]. CDK1 and RAB8A separately interact with
pericentriolar material 1 (PCM1) to control the assembly and disassembly of the cilia [265].
In cluster II, ATM-interactor (ATMIN) is a transcriptional regulator of the dynein light
chain LC8-type 1 (DYNLL1) essential for normal lung morphogenesis and ciliogenesis [266,267].
The inactivation of the ATMIN transcriptional regulator showed a moderate but significant
reduction in the expression of a ciliogenic transcriptional regulator FOXJ1 and some IFT
protein-encoding loci, i.e., IFT40 (IFTA), IFT88, and IFT72 (IFTB) [266]. Goggolidou and
co-workers also demonstrated the presence of bulges at the cilia base in both DYNLL1
and DYNC2H1 (component of mammalian dynein 2) mutants, which is a characteristic
phenotype representing defective cytoplasmic dynein 2 functions [266]. The ATMIN
mutation also affected the hedgehog signaling [266], which needs normal cilia to function in
lungs [268]. It has already been reported that the lungs with defective hedgehog signaling
could lead to pulmonary defects [269,270]. Moreover, impaired retrograde IFT due to
mutations in the cytoplasmic dynein 2 heavy chain, DYNC2H1, and the IFT-A genes IFT122,
WDR35/IFT121, and IFT43 have also been reported to cause lung mis-patterning [271].
Cluster III shows an interaction between MCIDAS and FOXJ1, two of the important fac-
tors that regulate ciliation in multi-ciliated cells (Figure 4a). In a conserved transcriptional
cascade, the inhibition of Notch signaling induces MCIDAS followed by the establish-
ment of a ternary complex with E2F-4/5 and Dp1, which further activates downstream
ciliary transcription factors, including RFX2 and FOXJ1, and controls the expression of core
multi-ciliogenesis genes [18,19,71,72].
Biology 2021, 10, 95 19 of 37

Figure 4. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-induced changes in the molecular machinery
regulating mucociliary clearance. (a) Interactome representing the SARS-CoV-2 infection-induced implications in the
molecular machinery regulating mucociliary clearance. The principal nodes and paths driving the molecular network were
analyzed using Cytoscape 3.7.2. The network analysis was performed using the betweenness centrality algorithm, where the
sizes of the nodes (circles) indicate betweenness. The color scale ranges from blue–green–yellow to orange, representing low
to high betweenness values, respectively. Clustering analysis was performed by employing the MCL algorithm. Perforated
shapes represent different clusters (I–VIII). Clusters I–V consist of proteins involved in cell cycle regulation, IFT machinery,
cilia biogenesis and regulation, cAMP regulation, and immune defense, respectively. Clusters VI–VIII represent proteins
acting as SARS-CoV-2 entry point and infection targets, respectively. CDK1 (cluster I), minichromosome maintenance 3
(MCM3; cluster I), RAB8A (cluster II), CD209 (cluster V), angiotensin converting enzyme (ACE2; cluster VI), PPIA (cluster
VII), and neuropilin-1 (NRP1; cluster VIII) proteins are known to interact with viral proteins during the course of infection.
(b) Partial interactome of SARS-CoV-2 and host proteins extracted from Network Data Exchange (NDEx) public server
(www.ndexbio.org) representing the IntAct/IMEx Coronavirus Dataset. The protein–protein interaction networks (PPIs)
were visualized in Cytoscape 3.7.2 (with CyNDEx-2 application) with the Compound Spring Embedder (CoSE) layout. Each
edge represents interaction studied in different experiments. Edge highlighted in red represents evidence from mutation
study. Dashed lines represent spoke-expanded interactions. Node color shows different species origin, where pink and
orange colors represent SARS-CoV-2 origin and the blue color is used to denote human origin proteins.
Biology 2021, 10, 95 20 of 37

Furthermore, cluster IV revealed the interaction among the proteins involved in cAMP
regulation, i.e., adenylate cyclase (ADCY3 or AC3) and phosphodiesterases (PDE5A and
PDE4B) in cilia (Figure 4a). Ciliary beating is one of the crucial components for the accom-
plishment of an efficient mucociliary clearance whose malfunction causes several chronic
airway diseases such as chronic bronchitis and cystic fibrosis. The intraciliary cAMP along
with intracellular pH, Ca2+ , and HCO3 − concentration regulates ciliary beat frequency
(CBF) [272,273]. The cAMP-dependent activation of protein kinase A (PKA) causes the
phosphorylation of an outer arm dynein light chain, which speeds up micro tubular slid-
ing and CBF [274]. However, the involvement of adenylate cyclase in this process is not
fully understood. The HCO3 − sensitive adenylate cyclase and cAMP also regulate the
CFTR, which is crucial for innate defenses in the lung during exacerbations of airway
diseases [275]. During P. aeruginosa and K. pneumoniae infections of the airways, the expres-
sion of IL-17A has been shown to become elevated, which in turn increases the secretion
of HCO3 − in the airway lumen [276–278]. Jiang and co-workers also demonstrated the
mechanism behind the cAMP-based regulation of ciliary hedgehog (Hh) signaling, where
they reported that Gα(s)-coupled GPCR regulated ciliary cAMP negatively regulated Hh
transcriptional activity in cilium [279].
Mucus cell hyperplasia/metaplasia and the increased size/frequency of submucosal
gland causes an excessive production of mucus in asthma and COPD patients [280–283].
MUC5AC and MUC5B are the most abundantly found mucins in airway mucus secreted
during inflammatory lung diseases [284–286]. Inflammatory cytokines secreted into the
airways affected by lung diseases [287] are one of several stimuli that can promote mucin
genes’ expression. Inflammatory cytokines that can affect mucin expression are categorized
into pro-inflammatory (TNF-α and IL-1β), Th1 (IFN-γ), TH17 (IL-17A), and Th2 (IL-4, IL-9,
and IL-13) cytokines [30]. TNF-α and IL-1β promote MUC5AC expression via a variety of
mechanisms [288–292]. In one mechanism, TNF-α activates two different MAP kinases, i.e.,
ERK and p38, through distinct pathways that promote the downstream activation of cyclic
AMP-responsive element binding protein (CREB). The CREB protein further binds to a
CREB-responsive cis element present on the MUC5AC promoter and activates the expres-
sion of MUC5AC [289]. However, apart from the CREB-mediated activation of MUC5AC,
in NCI-H292 cells, TNF-α can also promote MUC5AC expression through NF-kB [293]. The
MUC5AC promoter also exhibits putative binding sites for NF-kB, which is considered a
major transcriptional regulator of MUC5AC gene expression [294]. IL-4, IL-9, and IL-13 are
also thought to affect the MUC5AC and MUC5B gene expression in airway epithelial cells,
though their roles are controversial [287,295,296]. IL-4 and IL-13 bind to a common alpha
receptor (IL-4Rα), activate Janus kinase (JAK) pathways followed by nuclear translocation
of STAT6, and the further activation of STAT6-responsive genes. Chen et al. (2003) demon-
strated that IL-6 and IL-17 can also upregulate both MUC5B and MUC5AC. In the primary
airway epithelial cells, IL-17A indirectly upregulates MUC5B expression, which is partially
dependent on the IL-6 and JAK2-dependent autocrine/paracrine loop. Furthermore, IL-6
controls the expression of MUC5B via the ERK MAP kinase pathway [295].
Cluster VI has an ACE2 protein that has been considered as an entry factor for SARS-
CoV-2 [216,218]. It has been documented that SARS-CoV infection causes a reduction
in cell membrane ACE2 concentration, and subsequently increases serum angiotensin II
(Ang II), which results in severe lung inflammation followed by the development of acute
respiratory distress syndrome (ARDS) [297]. Ang II produced by the action of ACE2 is a
vasoconstrictor but can also act as a pro-inflammatory cytokine via angiotensin receptor
type 1 (AT1R) [298]. The Ang II binding to AT1R activates NF-kB [298]. However, during
inflammation, STAT3 is essential for the complete activation of the NF-kB pathway [299].
STAT3 is stimulated by the IL-6 family of cytokines [299]. ARDS-associated SARS-CoV
infection is the result of the hyper-activation of the NF-kB pathway, which causes the
release of pro-inflammatory cytokines such as IL-6, TNF-α, and chemokines produced
by immune cells and non-immune cells. The SARS-CoV-dependent activation of NF-kB
followed the MyD88 pathway through pattern recognition receptors (PRRs) [300].
Biology 2021, 10, 95 21 of 37

Cluster VII showed the presence of another protein, i.e., cyclophilin A (PPIA), which
has a critical role in SARS-CoV-2 infection (Figure 4a). PPIA promotes the interaction be-
tween non-structural protein 1 (nsp1) of SARS-CoV and CD147, which causes a reduction
in the interferon responses in infected cells [301]. In lungs, a higher expression of CD147
was observed in both the epithelial tissues, as well as in innate and adaptive immune
cells (macrophages, monocytes, ILCs, NK cells, T cells, and B cells), suggesting them as
potential targets of infection via CD147 [302]. Cyclophilin/PPIA is also known to inhibit
NF-ATs (nuclear factor of activated T-cells) activation in T cells, which ultimately causes the
suppression of immune responses in infected cells [301,303]. Therefore, it was suggested
that cyclophilin–CD147 complex formation is an important event for the local and systemic
spread of SARS-CoV-2 via CD147 in a comparable way to TMPRSS2 and SLC6A19 for
ACE2. Cluster VII also possess a calcineurin subunit B type 1 (PPP3R1; regulatory subunit)
as an interacting partner (Figure 4a). Calcineurin is a Ca2+ and calmodulin regulated
serine/threonine-protein phosphatase that is well known for its role in signal transduc-
tion during T-cell activation [304]. The cyclophilin–cyclosporine A complex inhibits the
calcineurin-based dephosphorylation of NF-AT, which blocks T-cell-dependent cytokine
transcription (for example IL-2) and T-cell activation [305,306]. A C-type lectin, CD209,
was also observed in cluster V (Figure 4a), which mediates coronavirus infection by inter-
acting with the S glycoprotein’s RBD [229]. Cluster VIII possesses NRP1, an alternate entry
receptor for SARS-CoV-2, which helps in potentiating the viral infectivity [226,227]. NRP1
receptor binds to the sequence ‘RRAR’ at the S1 subunit’s C-terminus, and its expression
was found to be upregulated in patients with COVID-19 [228].
Furthermore, a partial interactome of SARS-CoV-2 and host proteins was extracted
from the Network Data Exchange (NDEx) public server (www.ndexbio.org) [307–309] repre-
senting the IntAct/IMEx Coronavirus Dataset [310] to understand the point of interactions
of SARS-CoV-2 proteins in the molecular machinery of mucociliary clearance (Figure 4b).
The extracted viral–host protein interaction network was visualized in Cytoscape 3.7.2
(with CyNDEx-2 application) with the Compound Spring Embedder (CoSE) layout (the
detailed methodology is present in the Supplementary Material, M&M S1). The viral–host
interactome showed the interaction of SARS-CoV-2 components with seven proteins, i.e.,
ACE2, PPIA, CDK1, MCM3, RAB8A, NRP1, and CD209, belonging to predicted molecular
machinery of mucociliary clearance (Figure 4b). The host ACE2 receptor protein behaves
as the entry point for SARS-CoV-2. For invading host target cells, the SARS-CoV-2 spike
(S) protein binds to an ACE2 receptor and is subsequently primed by a TMPRSS2. The
TMPRSS2, a type II transmembrane serine protease, further cleaves the S protein, resulting
in the fusion of viral and host lysosomal membranes [218]. The viral–host interactome also
showed a connection between spike protein (S), ACE2 receptor and heparin (Figure 4b).
Clausen and co-workers performed a docking analysis and demonstrated that the spike
protein, ACE2 receptor, and heparin form a ternary complex where heparin broadens the
RBD’s open conformation that associates with ACE2 [230]. Targeting the heparin/heparan
sulfate-mediated enhancement of binding to ACE2 could be considered as a potential
target for developing therapy for inhibiting viral adhesion. Though not revealed from the
viral–host interactome, an alternate mechanism for SARS-CoV-2 invasion has also been
deciphered recently in tissues with a low or absent ACE2 expression. The NRP1 host
receptor has been reported to bind to a polybasic sequence ‘RRAR’ located at the S1–S2
junction in the S glycoprotein that helps in SARS-CoV-2 entry [224,226,227]. As discussed
in the previous section, PPIA or cyclophilin is important for the local and systemic spread
of SARS-CoV-2 via CD147. PPIA is known to promote the interaction between SARS-CoV
nsp1 and CD147, which causes a reduction in the interferon responses in infected cells [301].
In the viral–host interactome, PPIA also interacts with NendoU (orf1ab polyprotein) and the
SARS-CoV-2 nucleocapsid (N) protein (Figure 4b). Luo and co-workers also demonstrated
the SARS-CoV nucleocapsid protein binding to human cyclophilin (hCypA or PPIA) with
a high affinity [311]. A hypothetical protein with an unknown function, named y14_sars2
(ORF14 of SARS-CoV-2) in the viral–host network, was found to interact with the CDK1
Biology 2021, 10, 95 22 of 37

and MCM3 (Figure 4b). CDK1 activity gets considerably diminished upon SARS-CoV-2
infection and causes an S/G2 phase arrest that supports viral replication by ensuring a
sufficient resource of nucleotides, as well as other essential host DNA repair/replication
proteins [312]. In addition, MCM3 also interacts with the non-structural proteins (nsp3,
nsp7, nsp8, nsp10, and RdRP) (Figure 4b). The viral non-structural proteins are cleaved
products of polyproteins ORF1a and ORF1ab, and they are involved in the formation
of viral replication/transcription complex (RTC). Virus infection upregulates the TGFβ
pathway, which modulates cell survival, motility, innate immune responses, and ultimately
leads to fibrosis (hallmarks of COVID-19) [313,314]. The viral–host network also showed
the interaction between ORF3a (ap3a_sars) and TGFBR2 (one of the TGFβ-associated fac-
tors) (Figure 4b). Stukalov and co-workers performed a network diffusion analysis and
showed the association between ORF3, TGFβ-associated factors (namely TGFB1, TGFB2,
LTBP1, TGFBR2, FURIN, and BAMBI), and the virus-mediated upregulation of fibrinogens,
fibronectin, SERPINE1, and integrin(s) [315]. As discussed earlier, CD209 can mediate coro-
navirus infection by interacting with the RBD of the S glycoprotein [229]. The viral–host
interactome also showed the CD209 interaction with the spike protein (Figure 4b).
The overall network has provided the probable pathway involved in the regulation
of mucociliary clearance and associated processes following SARS-CoV-2 infection. Host
proteins like ACE2, PPIA, NRP1, and CD209 act as the entry points and infection targets
upon SARS-CoV-2 invasion. The invading SARS-CoV-2 may further activate the host
immune system, which in turn activates mucins (MUC5AC and MUC5B) and causes
exessive mucus production, a condition commonly found in inflammatory repiratory
diseases. SARS-CoV-2 infection might influence the cAMP-dependent ciliary beat frequency
critical for an efficient mucociliary clearance. SARS-CoV-2 might also impact the host
proteins controlling the cell cycle (CDK1 and MCM3), IFT machinery (IFT88, IFT20, and
BBS1) and ciliogenesis (DYNLL1, DYNC2LL1, DYNC2H1, FOXJ1, RFX3, and MCIDAS).
Thus, based on the protein–protein network analysis (Figure 4a,b), we have predicted the
probable regulatory mechanism and the points of interactions for viral proteins in the
molecular machinery of host mucociliary clearance upon SARS-CoV-2 infection.

4.3. Modulation of Gene Expression of the MCC System upon SARS-CoV-2 Infection
The shotgun RNA sequencing profiles of nasopharyngeal swabs from SARS-CoV-2-
infected patients versus healthy individuals revealed the in vivo transcriptional response
to infection by the coronavirus. The R package DESeq 2 (Version 1.26.0) [316] was utilized
to calculate the normalized gene read counts and the differential expression (log2 fold
change) of mucociliary clearance related genes in SARS-CoV-2-infected and healthy indi-
viduals (detailed methodology is present in the Supplementary Material, M&M S2) using
NCBI GEO RNA-seq dataset published by Lieberman and co-workers (GEO accession:
GSE152075) [317]. Figure 5 depicts the differential expression of gene transcripts related
to the mucociliary clearance machinery. Most of the selected gene transcripts showed
differential expression (log2 fold change > 1), but the changes were not found to be sig-
nificant (padj > 0.05) in any case (Table S3). The expression data reported by Lieberman
et al. (2020) also revealed an unexpected downregulation of the MUC5AC gene and a few
inflammatory cytokines (IL-6 and TNF) in SARS-CoV-2-infected patients (Figure 5). These
unexpected observations can be explained by the fact that nasopharyngeal swabs from
patient and healthy individuals were collected for RNA-seq analysis, which is not an ideal
or sensitive anatomic location to test for systemic inflammation. Therefore, systematic and
detailed studies are required to understand the complete mechanism of the transcriptional
regulation of mucociliary clearance related genes in SARS-CoV-2-infected individuals,
which is a rather unexplored area till date.
Biology 2021, 10, 95 23 of 37

Figure 5. Differential expression (log2 fold change) of mucociliary clearance related gene transcripts in SARS-CoV-2-infected
patients relative to healthy individuals. Clusters I–V represent genes associated with cell cycle regulation, IFT machinery,
cilia biogenesis and regulation, cAMP regulation, and immune defense, respectively, whereas cluster VI and VII represent
genes controlling SARS-CoV-2 entry and infection, respectively.

5. Molecular Basis of Repurposing of Drugs for Mitigating SARS-CoV-2-Induced

Lung Cilia and MCC Dysfunctions
At this critical time of an ongoing pandemic, researchers worldwide have delved into
the development of an effective therapeutic agent to tackle COVID-19. Until an effective
antidote is developed, the repurposing of existing drugs seems to be the most viable
strategy to manage COVID-19. As SARS-CoV-2 affects the respiratory system and destroys
the ciliated cells, an investment in existing drugs that improve pulmonary function by
enhancing the mucociliary clearance system seems necessary. Bronchitol® (Pharmaxis Ltd.)
is an inhaled dried powder preparation of mannitol that is used as a management strategy
for adult patients diagnosed with cystic fibrosis [318]. It targets the lungs and acts by
increasing the osmolarity of the periciliary fluid that helps to hydrate the lung surface [319].
This augments mucociliary clearance and also improves cough clearance, thereby sloughing
off the mucus obstruction and preventing the associated pulmonary exacerbations seen
in cystic fibrosis. β-adrenergic agonists increase the ciliary beat frequency and improve
the mucociliary clearance rate in respiratory diseases like asthma and cystic fibrosis [320].
Davis and co-workers reported that terbutaline increased the active Cl− transport across
the canine tracheal airway epithelium [321]. This effects the passive movement of water
that hydrates the epithelial surface and may result in MCC augmentation. The recombinant
form of human DNase I (Pulmozyme® , dornase alfa) hydrolyses the DNA in the purulent
secretions from the airways of CF patients, reduces mucus viscoelasticity, and improves
pulmonary function [322,323]. The inhalation of amiloride (Na+ channel blocker) has
been found to improve sputum viscosity and mucus clearance, as well as to increase
cough-mediated sputum expectoration in CF patients, thus contributing to a delay in lung
function [324,325]. Denufosol, a P2Y(2) receptor agonist, functions by bypassing the CFTR
ion channels and activating alternate Cl− ion channels. This augments epithelial surface
Biology 2021, 10, 95 24 of 37

hydration, which is directly linked to an effective mucus clearance mediated by efficient

ciliary beating [326].

6. Concluding Remarks
The human respiratory system sustains life via the regular uptake of oxygen from the
environment. However, in the process, pollutants and noxious pathogens often invade
the lungs along with the vital oxygen. To evade and overcome such respiratory assaults,
many embedded guards like the cilia and the mucus lining exist in the MCC machinery.
Currently, we have been confronted by a challenge posed by the novel virus, SARS-CoV-2.
COVID-19 is an acute infectious respiratory disease that spreads via the nasal or oral route
and colonizes the respiratory system. As medical workers and scientists probe further
details of SARS-CoV-2 and the ensuing diseased state, people of elderly age and with
pre-existing health conditions have been recognized to be more vulnerable to infection and
to develop a severe form of the illness. The epithelial cilia in conjunction with respiratory
mucus are the primary barriers against such diseases that target the human airways.
Consequently, genetic or acquired aberrations in these defense arrangements like asthma,
cystic fibrosis, COPD, and ciliopathies like primary ciliary dyskinesia are perilous because
they provide a suitable environment for susceptibility to and fatality from COVID-19.
Thus, such impairments in the mucociliary escalator should be stringently identified and
managed, and surveillance should be intensified in the current scenario until an effective
antiviral intervention is developed.

Supplementary Materials: The following are available online at https://www.mdpi.com/2079-773

7/10/2/95/s1, Table S1: Table showing string output in terms of various evidence types (GO, local
network cluster, KEGG pathway, reactome pathways, annotated keywords and protein domains)
contributing to the molecular network of the lung cilia and MCC machinery regulating mucociliary
clearance in humans. The string output depicts the network statistics as well as the false discov-
ery rate (FDR) values for each evidence. The evidences with FDR value < 0.05 were considered
significant. Table S2: String output in terms of various evidence types (GO, local network cluster,
KEGG pathway, reactome pathways, annotated keywords and protein domains) contributing to the
network representing the SARS-CoV-2 infection induced implications in the molecular machinery
regulating mucociliary clearance. The string output depicts the network statistics as well as the false
discovery rate (FDR) values for each evidence. The evidences with FDR value < 0.05 were considered
significant. Table S3: List of differentially expressed transcripts related to mucociliary clearance
machinery in SARS-CoV-2 infected patients relative to healthy individuals.
Author Contributions: Conceptualization, S.K. and S.V.; methodology, M.S.K. and S.C.; formal
analysis, A. and M.S.K.; data curation, M.S.K. and S.C.; writing-original draft preparation, A. and
M.S.K.; writing-review and editing, A. and M.S.K.; visualization, A. and M.S.K.; supervision, S.K.
and S.V.; project administration, S.K.; funding acquisition, S.K. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by the Drugs and Pharmaceutical Research Programme (DPRP),
Government of India (grant number VI-D & P/546/2016-17/TDT (G) 07/03/2018) and DBT-IYBA
2016. The APC was funded by Professional Development Fund for S.K. M.S.K. was supported by
DSK-PDF of UGC, Government of India.
Institutional Review Board Statement: Ethical review and approval are not applicable, as it based
on bioinformatics analysis only.
Informed Consent Statement: Not applicable.
Data Availability Statement: Most of the data presented in this study are available in this article
and/or supplementary material. Restrictions may apply to the availability of some data as the same
was obtained from third party and are available from the authors/with the permission of third party
as referred in this manuscript.
Acknowledgments: We acknowledge all lab members for critical reading of the manuscript. S.C. is
thankful to Niraj Kumar Khemka (Quick is Cool, www.quickiscool.org) for all the help rendered.
Biology 2021, 10, 95 25 of 37

Conflicts of Interest: The authors declare no conflict of interest.

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